Deficiency of acid alpha glucosidase (GAA) results in a fatal infantile glycogen storage disorder, a juvenile onset or a late onset myopathy. I will complete the isolation of the cDNA and the gene for human GAA for use as probes to analyze DNA and RNA from normals and patients with infantile, juvenile and adult GAA deficiency. The analysis and use as a probe of the isolated gene for GAA will lead to better understanding of the apparent genetic heterogeneity in GAA deficiency. I. ISOLATION OF THE GENE FOR GAA I have isolated a 2Kb partial cDNA and a 6 Kb fragment of the genomic DNA for human GAA. To isolate the cDNA, I screened a gtll expression library with affinity purified polyclonal antibody to GAA and isolated a phage with a 2 Kb insert. This phage was recognized by a monoclonal antibody and hybridized specifically to human 17(17q21.2-q23) which I previously showed contains the gene for human GAA and to a mRNA of size(3.4 Kb) to code for the 110 Kd GAA protein. This mRNA is not detectable in cells from at least one patient with GAA deficiency. Based on the size of the GAA protein, I will isolate a complete cDNA by using labelled cDNA as probe to screen several different cDNA libraries. I will determine the complete size of the 5' untranslated region by primer extension. I have also previously used DNA mediated cotransformation to obtain a series of mouse 3T3 clones which express human GAA and contain very little human DNA. The isolated cDNA hybridizes to fragments in these "cotransformants" which comigrate with human DNA. I have constructed a genomic phage library with DNA from one cotransformant and isolated phage which contained exogenous DNA. One phage contained a 6 Kb fragment which hybridized to the cDNA and therefore contains a portion of the GAA gene. I will however isolate the complete gene using the cDNA to screen a genomic library of EcoR1-20 Kb fragments. II. STUDIES OF DNA AND RNA FROM HUMANS DEFICIENT FOR GAA The isolated genomic DNA or cDNA will be used to study mutant cells(a)initially to determine at the level of DNA, the presence or absence of all normal exons,(b) to determine the presence and quantitation of mRNA by Northern blot and by S1 nuclease analysis for normal splicing. Studies to more definitively study specific mutation would include direct cloning and sequencing of mRNA where produced and/or genomic DNA where mRNA is absent.